Qing-Fang Miao

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death. Targeted monotherapies against the actionable oncogenic drivers, including anaplastic lymphoma kinase (ALK) rearrangement and epidermal growth factor receptor (EGFR) dysregulation, are well-established for molecularly selected NSCLC patients. However, therapeutic resistance remains a major clinical challenge, and combinatorial strategies targeting distinct signaling pathways may overcome the limitations of single-agent targeted therapies. LR004-VC-MMAE, a novel EGFR-targeting antibody‒drug conjugate (ADC), shows potent antitumor efficacy in multiple EGFR-positive xenografts, yet its therapeutic potential in ALK-rearranged NSCLC remains largely unexplored. Here, we evaluated a novel combinatorial strategy using ALK inhibitors (crizotinib, ceritinib, alectinib, and lorlatinib) in combination with LR004-VC-MMAE in ALK-rearranged NSCLC models. We found that both ALK and cell-surface EGFR were highly expressed in ALK-rearranged NCI-H3122 and NCI-H2228 cell lines but were expressed at low levels in the ALK-wild-type NCI-H460 cell line. All ALK inhibitor-ADC regimens synergistically suppressed proliferation in ALK-rearranged NSCLC cells, while exerting little synergy in ALK wild-type and EGFR-low counterparts. Moreover, crizotinib or ceritinib combined with LR004-VC-MMAE induced significantly greater apoptosis in NCI-H3122 cells compared with either single agent. Mechanistically, the crizotinib-LR004-VC-MMAE combination markedly downregulated ALK and EGFR expression and triggered apoptosis via mitochondrial depolarization-mediated caspase cascade activation and endoplasmic reticulum stress-induced unfolded protein response. In NCI-H3122 xenografts, this combination achieved synergistic tumor inhibition without increased toxicity compared with either treatment alone. Collectively, our findings demonstrate the synergistic antitumor activity of ALK inhibitor plus LR004-VC-MMAE against ALK-rearranged, EGFR-high NSCLC, providing a mechanistic rationale for further clinical development of this combination strategy.

The immunosuppressive nature of tumor microenvironment (TME) limits the efficacy of current immunotherapies. Bispecific antibodies (bsAbs) offer a promising strategy to overcome this challenge by enabling localized immune activation. Specifically, simultaneously targeting the “don’t eat me” signal CD24 on tumor cells and the co-stimulatory receptor 4-1BB on immune cells may provide potent anti-tumor effects while mitigating the systemic toxicity typically associated with conventional 4-1BB agonists. Here, we present the preclinical evaluation of IBD0333, a novel bsAb targeting CD24 and 4-1BB. The binding affinity of IBD0333 to CD24 and 4-1BB was evaluated, and its pharmacodynamic effects were assessed through multiple in vitro assays. Anti-tumor efficacy of IBD0333 was evaluated using C57BL/6J-h4-1BB humanized mouse models in vivo, while pharmacokinetic and safety profiles were further investigated in cynomolgus monkeys. IBD0333 exhibited high-binding affinity to both human and cynomolgus monkey CD24 and 4-1BB proteins. Its agonistic activity toward 4-1BB was strictly dependent on CD24, as evidenced by potent NF-κB activation and cytokine induction only in the presence of CD24-positive tumor cells. IBD0333 also demonstrated appropriate FcγR binding and no-detectable antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity activity. In a syngeneic tumor model, complete tumor regression was achieved at doses of 1 and 3 mg/kg. Furthermore, IBD0333 was well tolerated in cynomolgus monkeys with no observed immunotoxicity or hepatotoxicity, and the no-observed-adverse-effect level following repeated administration was established as 120 mg/kg. IBD0333 is a tumor-conditionally activated anti-CD24×4-1BB bsAb that demonstrates potent antitumor efficacy along with a favorable safety and pharmacokinetic profile. These findings support its further clinical development.

Purpose CD30 serves as an ideal therapeutic target for lymphoma, but its variable expression and high relapse rate pose challenges in targeted therapy. This study aims to label the anti-CD30 monoclonal antibody with ⁶⁴Cu/¹⁷⁷Lu for immuno-positron emission tomography (immuno-PET) and radioimmunotherapy (RIT). Methods CD30 binding kinetics of anti-CD30-IgG (IMB16) were measured by Biolayer interferometry (BLI). Western blotting screened lymphoma cell lines for CD30 expression. Flow cytometry and immunofluorescence validated the specific binding of IMB16. IMB16 was conjugated to p-SCN-Bn-NOTA(NOTA) and p-SCN-Bn-DOTA(DOTA) for radiolabeling with ⁶⁴Cu and ¹⁷⁷Lu. [⁶⁴Cu]Cu-NOTA-IMB16 and [¹⁷⁷Lu]Lu-DOTA-IMB16 were used for immuno-PET and RIT in subcutaneous lymphoma NSG mouse models. Results IMB16 had a strong binding affinity to CD30 according to the BLI. Western blotting revealed high CD30 expression in Karpas299 cells and negative expression in Raji cells. Flow cytometry and immunofluorescence confirmed specific binding of IMB16 to CD30 on cell surface. Radiochemical purity of [⁶⁴Cu]Cu-NOTA-IMB16 and [¹⁷⁷Lu]Lu-DOTA-IMB16 exceeded 95%. In Immuno-PET imaging, CD30-positive Karpas299 tumours had a mean uptake value of 19.2 ± 0.9%ID/g (n = 3) at 24 h post-injection, significantly higher than Karpas299-blocked and Raji-negative groups (P < 0.001). A high radiation dose (300µCi) of [¹⁷⁷Lu]Lu-DOTA-IMB16 significantly inhibited tumour growth (80.2 ± 17.6% standardized tumour volume, n = 5) at 10 days post-injection, compared to controls. Ex vivo biodistribution and histological staining supported in vivo PET imaging and RIT results. Conclusions Labelling IMB16 with ⁶⁴Cu enabled non-invasive assessment of CD30 expression, while ¹⁷⁷Lu labelling effectively suppressed tumour growth in CD30-positive lymphoma. CD30-targeted theranostic show promise for patient stratification and treatment enhancement, warranting further clinical evaluation.

Trophoblast cell surface antigen 2 (Trop2) is considered to be an attractive therapeutic target in cancer treatments. We previously generated a new humanized anti-Trop2 antibody named hIMB1636, and designated it as an ideal targeting carrier for cancer therapy. Lidamycin (LDM) is a new antitumor antibiotic, containing an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). AE and LDP can be separated and reassembled, and the reassembled LDM possesses cytotoxicity similar to that of native LDM; this has made LDM attractive in the preparation of gene-engineering drugs. We herein firstly prepared a new fusion protein hIMB1636-LDP composed of hIMB1636 and LDP by genetic engineering. This construct showed potent binding activities to recombinant antigen with a K_D value of 4.57 nM, exhibited binding to Trop2-positive cancer cells and internalization and transport to lysosomes, and demonstrated powerful tumor-targeting ability in vivo. We then obtained the antibody-drug conjugate (ADC) hIMB1636-LDP-AE by molecular reconstitution. In vitro, hIMB1636-LDP-AE inhibited the proliferation, migration, and tumorsphere formation of tumor cells with half-maximal inhibitory concentration (IC₅₀) values at the sub-nanomolar level. Mechanistically, hIMB1636-LDP-AE induced apoptosis and cell-cycle arrest. In vivo, hIMB1636-LDP-AE also inhibited the growth of breast and lung cancers in xenograft models. Moreover, compared to sacituzumab govitecan, hIMB1636-LDP-AE showed more potent antitumor activity and significantly lower myelotoxicity in tumors with moderate Trop2 expression. This study fully revealed the potent antitumor efficacy of hIMB1636-LDP-AE, and also provided a new preparation method for LDM-based ADC, as well as a promising candidate for breast cancer and lung cancer therapeutics.

Herein, we first prepared a novel anti-TROP2 antibody-drug conjugate (ADC) hIMB1636-MMAE using hIMB1636 antibody chemically coupled to monomethyl auristatin E (MMAE) via a Valine-Citrulline linker and then reported its characteristics and antitumor activity. With a DAR of 3.92, it binds specifically to both recombinant antigen (K_D ∼ 0.687 nM) and cancer cells and could be internalized by target cells and selectively kill them with IC₅₀ values at nanomolar/subnanomolar levels by inducing apoptosis and G2/M phase arrest. hIMB1636-MMAE also inhibited cell migration, induced ADCC effects, and had bystander effects. It displayed significant tumor-targeting ability and excellent tumor-suppressive effects in vivo, resulting in 5/8 tumor elimination at 12 mg/kg in the T3M4 xenograft model or complete tumor disappearance at 10 mg/kg in BxPc-3 xenografts in nude mice. Its half-life in mice was about 87 h. These data suggested that hIMB1636-MMAE was a promising candidate for the treatment of pancreatic cancer with TROP2 overexpression.

Purpose Pancreatic cancer is a malignant tumor with a high degree of malignancy, strong heterogeneity, and high lethality. Trop2 is a transmembrane glycoprotein associated with the occurrence, development, and poor prognosis of pancreatic cancer. This study aims to develop ⁶⁴Cu/¹⁷⁷Lu-labeled anti-Trop2 monoclonal antibody (hIMB1636) for positron emission tomography (PET) imaging and radioimmunotherapy (RIT) application in pancreatic cancer tumor models. Methods The binding kinetics of hIMB1636 to Trop2 antigen was measured by Biolayer interferometry (BLI). Western blotting was used to screen the Trop2 expression of pancreatic cancer cell lines. Flow cytometry and cell immunofluorescence were used to evaluate the binding ability of hIMB1636 and Trop2 on the cell surface. hIMB1636 were conjugated with p-SCN-Bn-NOTA (NOTA) and DOTA-NHS-ester (DOTA) for ⁶⁴Cu and ¹⁷⁷Lu radiolabeling respectively. ImmunoPET imaging and RIT studies were performed using ⁶⁴Cu-NOTA-hIMB1636 and ¹⁷⁷Lu-DOTA-hIMB1636 in subcutaneous pancreatic cancer tumor models. Results hIMB1636 had a strong binding affinity to Trop2 according to the results of BLI. The T3M-4 cell line showed the strongest expression of Trop2 and specific binding ability of hIMB1636 according to the results of Western blotting, flow cytometry, and cell immunofluorescence. The radiochemical purity of ⁶⁴Cu-NOTA-hIMB1636 and ¹⁷⁷Lu-DOTA-hIMB1636 exceeded 95%. PET imaging showed gradually an accumulation of ⁶⁴Cu-NOTA-hIMB1636 in T3M-4 tumor models. The maximum tumor uptake was 8.95 ± 1.07%ID/g (n = 4) at 48 h post injection (p.i.), which had significant differences with T3M-4-blocked and PaTu8988-negative groups (P < 0.001). The high-¹⁷⁷Lu-hIMB1636 group demonstrated the strongest tumor suppression with standardized tumor volume about 94.24 ± 14.62% (n = 5) at 14 days p.i., significantly smaller than other groups (P < 0.05). Ex vivo biodistribution and histological staining verified the in vivo PET imaging and RIT results. Conclusions This study demonstrated that ⁶⁴Cu/¹⁷⁷Lu-labeled hIMB1636 could noninvasively evaluate the expression level of Trop2 and inhibit the Trop2-overexpressed tumor growth in pancreatic cancer tumor models. Further clinical evaluation and translation of Trop2-targeted drug may be of great help in the stratification and management of pancreatic cancer patients.